1. Field of the Invention
This invention relates to a bioluminescence reagent that the amount of luminescence may be maintained in a high level and moreover stably without decaying for a long time in a bioluminescence method, a method for quantitatively determining an adenosine phosphate ester using the reagent, and a method for quantitatively determining a substance taking part in an ATP conversion reaction system using the reagent.
The ATP conversion reaction system in the invention means any reaction system which is composed, for example of a combination of enzymes and substrates, and wherein ATP is formed or consumed at the time of reaction.
For example, it includes, so far known ATP assaying systems each utilizing a bioluminescence reaction (a reaction system wherein luciferase is made to act on ATP, luciferin, dissolved oxygen and magnesium ion to form AMP, pyrophosphoric acid, oxyluciferin, carbon dioxide and light), and an assaying system for an adenosine phosphate ester such as AMP is, ADP , ATP or cyclic AMP disclosed in the invention, and in addition a reaction system wherein another reaction system is combined with such an assay system (reaction system) and in the reaction system after the combination, ATP is formed or consumed at the time of reaction.
2. Description of the Related Prior Art
Heretofore, there has been known an ATP-quantitatively determining method by a so-called bioluminescent method wherein a bioluminescence reagent comprising luciferin, luciferase and magnesium ions (or other metallic ions) is reacted with a sample containing adenosine triphosphate (hereinafter referred to as ATP) and the amount of luminescence formed is quantitatively determined.
However, although this method has an advantage that ATP can be determined quickly, it has a disadvantage that the stability of luminescence is poor, and more specifically luminescence fades out in a very short time, and therefore it has a problem that for securing sensitivity and accuracy, it gets necessary to control the reaction time strictly and use a luminometer, a measuring apparatus equipped with an autoinjection function for capturing luminescence fading out in a short time.
Heretofore, some techniques for stabilizing luminescence over a long time in this bioluminescence method have been developed, but any of them has problems to be improved.
Namely, in a method which comprises adding coenzyme A (hereinafter abbreviated as CoA) to a reaction system (Patent Kohyo No. 6-500921), a luciferase-luciferin reaction is carried out in the presence of CoA, under a condition of lowering the peak intensity of light formed in the reaction, by a small amount (for example, by about 3 to 30% of the peak intensity under the absence of this condition), and thereby all the amount of light emitted in the reaction (i.e., amount measured by integrating the curve of intensity to time) is increased, and thereby all the light output can be measured more simply and more accurately.
However, in this method, it is compelled to lower the peak (sensitivity) of light formed by the reaction, and the relations between the intensity of luminescence and time get to be curve relations, and the amount of luminescence gradually decays. Therefore, it cannot be expected to maintain the amount of luminescence at a high level stably for a long time.
Further, since a thiol reagent has a SH group on the structure, it, in general, is easily oxidized and has a problem on storage as a solution. Further, since CoA on the market is one extracted from a yeast or the like, it is expensive, and moreover a possibility that ATP, etc. are mixed is noted. The inclusion of ATP is not desired because it causes the increase of the background when ATP is an object to be assayed.
In a method which comprises adding pyrophosphoric acid to the reaction system at the time when a bioluminescence reaction progressed in some extent and the amount of luminescence started to lower, and thereby forming intense luminescence momentarily (Arch. Biochem. Biophys. 46, 399-416; 1955), the peak intensity of light is increased again in the middle of the luminescence reaction and thereby the extension of the luminescence time is attempted, but it is impossible to stabilize luminescence over a long time.
In a method which comprises using, in a bioluminescence method, a D-luciferin analog in the reaction system as a competitive inhibitor (Patent Kokai No. 55-13893), although the amount of luminescence can be maintained at a certain level and stably for a long time, the intensity of luminescence is suppressed or inhibited by at least 25%, particularly 50 to 90% by the addition of the inhibitor, and thus the method has a drawback that the lowering of sensitivity is compelled.
In a method which comprises using a polyphosphoric acid compound and a sulfhydryl compound together in a bioluminescence method (Patent Kokai No. 8-47399), the peak intensity of light formed in the luminescence reaction is increased and thereby the extension of the luminescence time is attempted, but it is impossible to maintain luminescence stably over a long time.
Further, as to the above bioluminescence methods, there is no regeneration of ATP in any of them, and they each have a drawback that luminescence decays with time lapse as ATP is consumed.
Therefore, for maintaining the amount of luminescence stably for a long time without decaying, it is compelled to examine the substrate concentrations, enzyme concentrations, pH, temperature, addition of a suppressor or inhibitor, etc., and even thereby sufficiently satisfactory results cannot be expected.
On the other hand, a quantitative determination method of cyclic AMP using a bioluminescence method shown by the following reaction formula is known. ##STR1##
This method is characterized by a method of quantitatively determining cyclic AMP by Reaction 1 wherein cyclic AMP is hydrolyzed with cyclic-3',5'-nucleotide phosphodiesterase to form AMP in the reaction system, Reaction 2 wherein the AMP is reacted with adenylate kinase in the presence of magnesium ion and a trace amount of ATP to convert it to ADP, Reaction 3 wherein the ADP is reacted with pyruvate kinase in the presence of magnesium ion and phosphoenolpyruvic acid to convert it to ATP and pyruvic acid, Reaction 4 wherein the ATP is reacted with luciferase in the presence of luciferin, magnesium ion (or other metallic ions) and dissolved oxygen to form luminescence, and measuring the amount of luminescence formed in Reaction 4 (METHODS IN ENZYMOLOGY 38, 62-65; 1974).
However, in this method, AMP formed from cyclic AMP is converted to ADP by the reaction with adenylate kinase in the presence of a trace amount of ATP, and the ADP is converted to ATP with pyruvate kinase, and therefore, it is required to add a trace amount of ATP in advance to the reaction system.
This additions of ATP is not desirable because when cyclic AMP is the object to be assayed, the ATP causes the increase of the background.
As a result, a blank value corresponding to cyclic AMP of at least 1.3.times.10.sup.-9 M is formed, and thus the method has a problem that cyclic AMP cannot be determined with high sensitivity.
Thus the invention aims to provide a bioluminescence reagent that the amount of luminescence is maintained in a high level and moreover stably without decaying for a long time in a bioluminescence reaction, and provide a method for quantitatively determining an adenosine phosphate ester or a substance taking part in an ATP conversion reaction in high sensitivity and high accuracy using an inexpensive and simple measuring apparatus.